The PI3K/ERK Dual Inhibitor AEZS-136 Induces ROS-Dependent Necroptotic Cell Death and Exerts Potent Antitumor Effects In NOD/SCID Mice With Hodgkin Lymphoma Cell Line Xenografts

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3067-3067
Author(s):  
Silvia L Locatelli ◽  
Giuliano G Stirparo ◽  
Silvia Tartari ◽  
Elena Saba ◽  
Luca Rubino ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
S Phase ◽  
Scid Mice ◽  
Ros Generation ◽  
Oxygen Species ◽  
Dual Inhibitor ◽  
Dose Dependent

Abstract Introduction Disease relapse and resistance to chemotherapy represent challenging issues for Hodgkin Lymphoma (HL) patients. PI3K/AKT and RAF/MEK/ERK pathways are constitutively activated in the majority of HL patients, thus representing attractive therapeutic targets. Previous results from our phase II study indicate that combining the PI3K/AKT inhibitor perifosine with the RAF/MEK/ERK inhibitor sorafenib can achieve significant clinical responses in relapsed/refractory HL. The present study was therefore aimed at characterizing the in vitro and in vivo activity and mechanism(s) of action of a novel PI3K/ERK dual inhibitor AEZS-136 (Æterna Zentaris GmbH, Germany, EU). Methods Four HL cell lines (L-540, SUP-HD1, KM-H2 and L-428) were used to investigate the in vitro effects of AEZS-136 on cell growth, cell cycle distribution, gene expression profiling (GEP), and apoptosis. Live cell imaging experiments were performed to asses the production of reactive oxygen species (ROS). Western blotting (WB) was used to assess the effects of AEZS-136 on MAPK and PI3K/AKT pathways as well as apoptosis. The antitumor efficacy of AEZS-136 was investigated in vivo in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Results Exposure of L-540, SUP-HD1, KM-H2 and L-428 cell lines to AEZS-136 induced a marked, early and time-dependent dephosphorylation of PI3K/Akt and MAPK pathways that was associated with a significant time and dose-dependent cell growth inhibition [80 ± 3% (mean ±SEM) in the L-540 and SUP-HD1 responsive cell lines] and S phase cell cycle arrest. Indeed, upon AEZS-136 treatment the mean (±SEM) percentages of cells in S phase were reduced by 3-fold (13 ± 1%) as compared to control (33 ± 2%). Significant levels of cell death, as assessed by AnnexinV/PI staining, were only observed in L-540 (62 ± 9 vs 14 ± 3%, P ≤.0001) and SUP-HD1 (46 ± 2% vs 15 ± 2%, P ≤.0001) cell lines and were associated with severe mitochondrial dysfunction (up to 40%, P ≤.001). While no activation of caspase-3 and PARP cleavage were observed in L-540 and SUP-HD1 cells treated with AEZS-136, a potent generation of reactive oxygen species (ROS) was observed upon AEZS-136 treatment (up to 90%, P≤.0001). Pretreating cells with the ROS inhibitor YCG063 strongly inhibited AEZS-136-induced ROS generation, mitochondrial dysfunction and cell death, whereas the pan-caspase inhibitor Z-VADfmk did not. Since ROS generation has been implicated in mediating necroptosis, we tested if blocking programmed necrosis with Necrostatin-1 could prevent AEZS-136-induced cytotoxicity. When L-540 cells were treated with AEZS-136 in the presence of Necrostatin-1, cell death and ROS generation were completely prevented, suggesting that cell death was mechanistically related to necroptosis. Additionally, HL cells responsive to AEZS-136-induced cell death showed a pronounced JNK activation whose inhibition by the JNK inhibitor SP600125 reduced cell death and ROS generation. Furthermore, AEZS-136-increased JNK phosphorylation was inhibited by Necrostatin-1 or YCG063, suggesting that ROS-dependent necroptosis was linked to JNK. Interestingly, GEP analysis of L-540 and SUP-HD1 cell lines, but not KM-H2 and L-428 cells, indicated that AEZS-136 treatment induced upregulation of genes involved in positive regulation of cell death. In addition, in KM-H2 and L-428 cells, AEZS-136 strikingly induced the expression of the immediate early response 3 (IER3). Silencing of IER3 restored sensitivity of KM-H2 and L-428 cells to AEZS-136-induced necroptotic cell death, suggesting that IER3 acts as the signaling molecule that mediated AEZS-136-resistance to oxidative cell death. Finally, in vivo experiments were conducted to investigate the antitumor activity of AEZS-136. Treatment of NOD/SCID mice bearing L540 tumor nodules with increasing dose of AEZS-136 (30 – 60 mg/Kg body weight, PO, 5 days/2 weeks) resulted in a dose-dependent reduction of tumor growth (mean TGI of 70%, P ≤.0001) compared to vehicle-treated controls. No mice experienced any apparent treatment-related toxicity. Conclusions The PI3K/ERK dual inhibitor AEZS-136 demonstrates a potent antitumor activity against HL cell lines by targeting aberrant expression of MAPK and PI3K/Akt pathways. These data support further clinical evaluation of AEZS-136 in refractory/relapsed HL patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Combining IMGN779, a Novel Anti-CD33 Antibody-Drug Conjugate (ADC), with the PARP Inhibitor, Olaparib, Results in Enhanced Anti-Tumor Activity in Preclinical Acute Myeloid Leukemia (AML) Models

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1645-1645 ◽  
Author(s):  
Scott Portwood ◽  
Robert A Puchalski ◽  
Russell M Walker ◽  
Eunice S. Wang
Keyword(s):  
Cell Lines ◽  
Combination Treatment ◽  
Parp Inhibitor ◽  
Single Agent ◽  
S Phase ◽  
Complex Karyotype ◽  
Anti Tumor Activity ◽  
Dose Dependent

Abstract Background: New therapies for AML are urgently needed. IMGN779 is a novel CD33-targeting ADC that utilizes as the cytotoxic agent a DNA-alkylatingindolinobenzodiazepine, DGN462. Previous data have shown that IMGN779 exerts dose-dependent activity against human CD33+ AML cells in vitro and in vivo. We hypothesized that combination treatment of AML cells with the poly (ADP-ribose) polymerase (PARP) inhibitor, olaparib, which blocks cellular DNA repair would further enhance the anti-leukemic activity of IMGN779 in preclinical human AML models. Methods:Human CD33+ AML cell lines (HEL, MV4-11, HL60) were treated in vitro with controls, IMGN779, olaparib, and IMGN779 + olaparib. Proliferation was measured by WST-8 reagent. Synergistic/additive effects were calculated using Compusyn software. Flow cytometry was performed to assess apoptosis, viability, and cell cycle effects. SCID mice were engrafted systemically with human AML (HEL-luciferase) cells followed by treatment with IMGN779, olaparib, or both drugs in combination. Changes in disease burden and possible treatment-related toxicities were determined by whole animal bioluminescent imaging, body weights, and time to morbidity, respectively. Primary cells from patients with relapsed/refractory AML characterized by complex karyotype or FLT-3 mutations were plated in methocellulose media supplemented with hematopoietic cytokines for colony formation unit assays (CFU). Vehicle, IMGN779 and/or olaparib in triplicate were added followed by quantification of leukemic CFU 15 days later with a Spot-RT3 camera mounted to an inverted microscope. Results: IMGN779 treatment induced significant growth inhibition in vitro in all CD33+ human AML cell lines tested that was dose dependent. IMGN779 cell killing was CD33 dependent. Olaparib induced cell death in human AML cell lines via reversal of DNA damage repair mechanisms. Combination treatment with IMGN779 (500 pM-1 nM) and olaparib (10-50 μM) significantly enhanced anti-leukemic effects over monotherapy in the same cell lines (representative data in Table 1). Combination indices for IMGN779 and olaparib therapy ranged from 0.7-0.9, consistent with synergistic effects. The combination markedly reduced overall cell viability, increased apoptosis, and induced almost complete S-phase cell cycle arrest as compared with controls and single-agent treatments. Exposure to a combination of IMGN779 and olaparib also significantly inhibited CFU growth of progenitor cells established from bone marrow samples of patients (n=7) with relapsed/refractory, FLT3-ITD, and/or complex karyotype AML. Statistically significant inhibition of viable CFUs was observed following combination IMGN779 (10 pM) and olaparib (1 μM) therapy as compared with monotherapy or vehicle controls (p<0.001). In vivo, IMGN779 administered as a single dose that ranged from 0.5 to 5 mg/kg, by antibody, was overall well tolerated in SCID mice bearing systemic human CD33+ AML (HEL-luciferase) xenografts. Significant dose-dependent anti-leukemic activity, as reflected by decreased leukemia burden and prolonged overall survival, was observed. Combination treatment with IMGN779 (3 mg/kg) and olaparib (100 mg/kg) further significantly decreased leukemic burden when compared with vehicle (p<0.0001), IMGN779 alone (p<0.01) and olaparib alone (p<0.05) on day 14 after dosing (Figure 1). Conclusion:The combination of the CD33-targeting ADC, IMGN779, and the PARP inhibitor, olaparib, enhanced anti-tumor activity in multiple preclinical human CD33+ AML models. The combination increased in vitro DNA damage, apoptosis, S-phase arrest, and cell death effects on human CD33+ AML cells vs. single agent therapy. Combination therapy also markedly inhibited colony formation of primary AML cells representing clinically chemoresistant disease and significantly decreased in vivo leukemic burden in systemic human AML xenograft models. Our results support the future clinical investigation of this novel combinatorial regimen for AML therapy. In vivo IMGN779 + olaparib treatment in systemic HEL AML xenografts In vivo IMGN779 + olaparib treatment in systemic HEL AML xenografts Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Walker: ImmunoGen, Inc.: Employment.

The histone Deacetylase Inhibitor Givinostat in Combination with Sorafenib Induces Reactive Oxygen Species (ROS) Generation and Exerts Potent Antitumor Effects in NOD/SCID Mice with Hodgkin Lymphoma Cell Line Xenografts

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3711-3711
Author(s):  
Silvia L Locatelli ◽  
Anna Guidetti ◽  
Loredana Cleris ◽  
Silvia Tartari ◽  
Alessandro M. Gianni ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Cell Lines ◽  
Drug Combination ◽  
Fold Increase ◽  
Time Dependent ◽  
Ros Generation ◽  
Oxygen Species ◽  
Sorafenib Treatment

Abstract Abstract 3711 INTRODUCTION: Patients with refractory or relapsed classical Hodgkin Lymphoma (cHL) represent an unmet medical need and would benefit from the development of new therapies. Histone deacetylases (HDACs) and the RAF/MEK/ERK pathway are aberrantly controlled in cHL and influence a broad repertoire of tumor processes, suggesting a rationale for therapeutically targeting these pathways. We targeted these pathways using the HDAC inhibitor Givinostat (Italfarmaco S.p.A., Milan, Italy), and the RAF/MEK/ERK inhibitor Sorafenib (Nexavar, Bayer, Germany, EU) in order to investigate in vitro and in vivo the activity and mechanism(s) of action of this two-drug combination. METHODS: Three cHL cell lines, including HDLM-2, L-540 and HD-MyZ, were used to investigate the effects of Givinostat and Sorafenib, used alone or in combination, by means of in vitro assays evaluating cell growth and cell survival. Additionally, live cell imaging was used to asses the production of reactive oxygen species (ROS), and Western blotting (WB) to assess modulating effects of the two-drug combination on MAPK, PI3K/AKT, HDACs as well as the apoptotic pathways. The efficacy of Givinostat/Sorafenib combination was finally confirmed in NOD/SCID mice with cHL cell line xenografts. RESULTS: While Givinostat and Sorafenib as single agents exerted a limited activity against cHL cells, the combined Givinostat/Sorafenib treatment was associated with potent dephosphorylation of MAPK and PI3K/Akt pathways and significantly increased H3 and H4 acetylation due to a nearly complete inhibition of class I and II HDACs. Furthermore, these events were associated with a time-dependent synergistic cell growth inhibition (70% to 90%) in all Givinostat/Sorafenib-treated cHL cells. Upon Givinostat/Sorafenib exposure, HDLM-2 and L-540 cell lines showed significantly (P ≤.0001) increased levels of apoptosis (90 ± 2% and 96 ± 1%, respectively) and mitochondrial dysfunction (up to 70%, P≤.0001), as compared with single agents. Apoptosis induced by Givinostat/Sorafenib combination failed to induce processing of caspase-8, −9, −3, or cleavage of PARP, and was not reversed by the pan-caspase inhibitor Z-VADfmk, suggesting the occurrence of caspase-independent apoptosis. Besides downregulating the expression of the anti-apoptotic protein Mcl-1 and ERK1/2 phosphorylation, Givinostat/Sorafenib strongly increased expression of the BH-3 only protein Bim, compared to single treatments. These findings were dependent on a potent, early and time-dependent ROS generation (up to 60%, P≤.0001) that was synergistically induced by Givinostat/Sorafenib treatment. Additionally, pretreatment of cHL cells with the ROS inhibitor YCG063 prevented the generation of ROS as well as mitochondrial membrane depolarization along with cell death induced by the two-drug combination, suggesting that ROS generation is the triggering event in Givinostat/Sorafenib induced-cell death. In vivo Givinostat/Sorafenib treatment significantly reduced the growth of L-540 and HD-MyZ nodules, resulting in an average 35% to 65% tumor growth inhibition (P ≤.0001) compared to single treatments, in the absence of any toxicity. Interestingly, as compared to controls or treatment with single agents, the combined Givinostat/Sorafenib treatment significantly increased in vivo Bim expression (7- to 21-fold increase, P ≤.0001), resulting in a marked tumor necrosis (3- to 5-fold increase, P ≤.0001). CONCLUSIONS: The combined Givinostat/Sorafenib treatment demonstrates a potent preclinical in vitro and in vivo activity against cHL cell lines by targeting aberrant expression of HDACs and MAPK. Antitumor activity of this combination involves ROS generation and Bim upregulation and provides a rationale for clinical studies using this combination in refractory/relapsed cHL patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Mitochondrion-Dependent Cell Death in TDP-43 Proteinopathies

Biomedicines ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 376
Author(s):  
Chantal B. Lucini ◽  
Ralf J. Braun
Keyword(s):  
Cell Death ◽  
Oxygen Species ◽  
In Vivo Models ◽  
Dependent Cell ◽  
Cell Death Mechanisms ◽  
Sting Pathway ◽  
Mitochondrial Membrane Permeabilization

In the last decade, pieces of evidence for TDP-43-mediated mitochondrial dysfunction in neurodegenerative diseases have accumulated. In patient samples, in vitro and in vivo models have shown mitochondrial accumulation of TDP-43, concomitantly with hallmarks of mitochondrial destabilization, such as increased production of reactive oxygen species (ROS), reduced level of oxidative phosphorylation (OXPHOS), and mitochondrial membrane permeabilization. Incidences of TDP-43-dependent cell death, which depends on mitochondrial DNA (mtDNA) content, is increased upon ageing. However, the molecular pathways behind mitochondrion-dependent cell death in TDP-43 proteinopathies remained unclear. In this review, we discuss the role of TDP-43 in mitochondria, as well as in mitochondrion-dependent cell death. This review includes the recent discovery of the TDP-43-dependent activation of the innate immunity cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway. Unravelling cell death mechanisms upon TDP-43 accumulation in mitochondria may open up new opportunities in TDP-43 proteinopathy research.

scholarly journals TB-4 Antitumor effects of a novel curcumin derivative curcumin monoglucuronide on glioblastoma cells in vitro and in vivo

2021 ◽  
Vol 3 (Supplement_6) ◽  
pp. vi6-vi6
Author(s):  
Takashi Fujii ◽  
Shun Yamamuro ◽  
Masamichi Takahashi ◽  
Akihide Kondo ◽  
Yoshitaka Narita ◽  
...  
Keyword(s):  
Cell Death ◽  
Water Soluble ◽  
Dependent Manner ◽  
Antitumor Effects ◽  
Multiple Cell ◽  
Human Gbm ◽  
Dose Dependent ◽  
Novel Therapeutic

Abstract The therapeutic outcome of glioblastomas (GBMs) is still very poor. Therefore, invention of novel therapeutic methods against GBM cases is considered urgent. The antitumor effects of naturally-derived compounds are attracting attention recently, and therapeutic efficacy of curcumin, a plant-derived compound previously used for multiple purpose, has been indicated in many cancer systems; however, clinical application of curcumin is considered difficult because of its poor bioavailability (under 1 %). Curcumin monoglucuronide (CMG), a water-soluble prodrug of curcumin recently developed for overcoming this weakness, has been demonstrated excellent antitumor effects for several malignancies in vitro and in vivo; therefore, we investigated the effects of CMG against GBM cells. CMG induced cell death of human GBM cells lines (T98G, U251MG, and U87MG) by dose dependent manner by triggering multiple forms of cell death such as apoptosis and perthanatos. Immunoblotting of CMG-treated GBM cell lysates demonstrated activation of multiple cell death signaling. Furthermore, immunodeficiency mice harboring intracerebral U87MG cell xenografts systemically treated by CMG showed significantly prolonged survival compared with control mice. These results suggest CMG would be a novel therapeutic agent against GBM cases.

scholarly journals Therapeutic evaluation of palbociclib and its compatibility with other chemotherapies for primary and recurrent nasopharyngeal carcinoma

2020 ◽  
Author(s):  
zhichao xue ◽  
Vivian Wai Yan Lui ◽  
Yongshu Li ◽  
Jia Lin ◽  
Chanping You ◽  
...  
Keyword(s):  
Cell Death ◽  
Nasopharyngeal Carcinoma ◽  
Cell Lines ◽  
Chemotherapeutic Drugs ◽  
Induce Cell Cycle Arrest ◽  
Ki 67 ◽  
Concurrent Treatment ◽  
Xenograft Models

Abstract Background: Recent genomic analyses revealed that druggable molecule targets were detectable in approximately 6% of patients with nasopharyngeal carcinoma (NPC). However, a dependency on dysregulated CDK4/6–cyclinD1 pathway signaling is an essential event in the pathogenesis of NPC. In this study, we aimed to evaluate the therapeutic efficacy of a specific CDK4/6 inhibitor, palbociclib, and its compatibility with other chemotherapeutic drugs for the treatment of NPC by using newly established xenograft models and cell lines derived from primary, recurrent, and metastatic NPC. Methods: We evaluated the efficacies of palbociclib monotherapy and concurrent treatment with palbociclib and cisplatin or suberanilohydroxamic acid (SAHA) in NPC cell lines and xenograft models. RNA sequencing was then used to profile the drug response–related pathways. Palbociclib-resistant NPC cell lines were established to determine the potential use of cisplatin as a second-line treatment after the development of palbociclib resistance. We further examined the efficacy of palbociclib treatment against cisplatin-resistant NPC cells. Results: In NPC cells, palbociclib monotherapy was confirmed to induce cell cycle arrest in the G1 phase in vitro . Palbociclib monotherapy also had significant inhibitory effects in all six tested NPC tumor models in vivo , as indicated by substantial reductions in the total tumor volumes and in Ki-67 proliferation marker expression. In NPC cells, concurrent palbociclib treatment mitigated the cytotoxic effect of cisplatin in vitro . Notably, concurrent treatment with palbociclib and SAHA synergistically promoted NPC cell death both in vitro and in vivo . This combination also further inhibited tumor growth by inducing autophagy-associated cell death. NPC cell lines with induced palbociclib or cisplatin resistance remained sensitive to treatment with cisplatin or palbociclib, respectively. Conclusions: Our study findings provide essential support for the use of palbociclib as an alternative therapy for NPC and increase awareness of the effective timing of palbociclib administration with other chemotherapeutic drugs. Our results provide a foundation for the design of first-in-human clinical trials of palbociclib regimens in patients with NPC.

scholarly journals Anticancer Activity of Cynomorium coccineum

Cancers ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 354 ◽  
Author(s):  
Mouna Sdiri ◽  
Xiangmin Li ◽  
William Du ◽  
Safia El-Bok ◽  
Yi-Zhen Xie ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Anticancer Activity ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cell Cycle Progression ◽  
Cycle Progression

The extensive applications of Cynomorium species and their rich bioactive secondary metabolites have inspired many pharmacological investigations. Previous research has been conducted to examine the biological activities and numerous interesting pharmaceutical activities have been reported. However, the antitumor activities of these species are unclear. To understand the potential anticancer activity, we screened Cynomorium coccineum and Cynomorium songaricum using three different extracts of each species. In this study, the selected extracts were evaluated for their ability to decrease survival rates of five different cancer cell lines. We compared the cytotoxicity of the three different extracts to the anticancer drug vinblastine and one of the most well-known medicinal mushrooms Amaurederma rude. We found that the water and alcohol extracts of C. coccineum at the very low concentrations possessed very high capacity in decreasing the cancer cells viability with a potential inhibition of tumorigenesis. Based on these primitive data, we subsequently tested the ethanol and the water extracts of C. coccineum, respectively in in vitro and in vivo assays. Cell cycle progression and induction of programmed cell death were investigated at both biological and molecular levels to understand the mechanism of the antitumor inhibitory action of the C. coccineum. The in vitro experiments showed that the treated cancer cells formed fewer and smaller colonies than the untreated cells. Cell cycle progression was inhibited, and the ethanol extract of C. coccineum at a low concentration induced accumulation of cells in the G1 phase. We also found that the C. coccineum’s extracts suppressed viability of two murine cancer cell lines. In the in vivo experiments, we injected mice with murine cancer cell line B16, followed by peritoneal injection of the water extract. The treatment prolonged mouse survival significantly. The tumors grew at a slower rate than the control. Down-regulation of c-myc expression appeared to be associated with these effects. Further investigation showed that treatment with C. coccineum induced the overexpression of the tumor suppressor Foxo3 and other molecules involved in inducing autophagy. These results showed that the C. coccineum extract exerts its antiproliferative activity through the induction of cell death pathway. Thus, the Cynomorium plants appear to be a promising source of new antineoplastic compounds.

scholarly journals IMMU-14. ONCOLYTIC VIRUS EXPRESSING A POSITIVE IMMUNE CHECKPOINT MODULATOR AS A THERAPEUTIC APPROACH FOR DIPG

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi122-vi122
Author(s):  
Virginia Laspidea ◽  
Montse Puigdelloses ◽  
Ignacio Iñigo-Marco ◽  
Marc Garcia-Moure ◽  
Iker Ausejo ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Cell Death ◽  
Cell Lines ◽  
Oncolytic Virus ◽  
Murine Models ◽  
Antitumoral Effect ◽  
Infected Cells

Abstract Diffuse intrinsic pontine glioma (DIPG) is an aggressive brain tumor, being the leading cause of pediatric death caused by cancer. We previously showed that administration of the oncolytic virus Delta-24-RGD to DIPG murine models was safe and led to an increase in the median survival of these animals. However, not all the animals responded, underscoring the need to improve this therapy. In order to increase the antitumoral effect of the virus, we have engineered Delta-24-RGD with the costimulatory ligand 4-1BBL (Delta24-ACT). 4-1BB is a costimulatory receptor that promotes the survival and expansion of activated T cells, and the generation and maintenance of memory CD8+ T cells. In this project, we evaluated the oncolytic effect of Delta24-ACT and the antitumor immune response in DIPG murine models. In vitro, Delta24-ACT was able to infect and induce cell death in a dose-dependent manner in murine DIPG cell lines. In addition, Delta24-ACT was able to replicate in these tumor cells and to express viral proteins. Moreover, infected cells expressed 41BBL in their membranes. Delta24-ACT could induce immunogenic cell death due to an increased secretion of ATP and calreticulin translocation to the membrane of infected cells (in no-infected cells it located in the ER), DAMPs that can trigger the immune response activation. In vivo, Delta24-ACT demonstrated to be safe in all the tested doses and was able to induce a significant increase in the median survival of the treated animals. Moreover, long-term survivors display immunological memory. Delta24-ACT treatment led to antitumoral effect in DIPG murine cell lines in vitro. Of significance, we have demonstrated that in vivo administration of Delta24-ACT is safe and results in an enhanced antitumor effect. Future in vivo studies will explore the underlying immune mechanism of the virus.

scholarly journals P06.01 Delta24-ACT oncolytic adenovirus as a therapeutic approach for DIPG

2019 ◽  
Vol 21 (Supplement_3) ◽  
pp. iii36-iii36
Author(s):  
V Laspidea ◽  
M Puigdelloses ◽  
M García-Moure ◽  
I Iñigo-Marco ◽  
J Gallego ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Cell Lines ◽  
In Vivo Studies ◽  
Immunogenic Cell Death ◽  
Murine Models ◽  
Antitumoral Effect ◽  
Infected Cells

Abstract BACKGROUND Diffuse intrinsic pontine glioma (DIPG) is an aggressive brain tumor, being the leading cause of pediatric death caused by cancer. We previously showed that administration of the oncolytic virus Delta-24-RGD to DIPG murine models was safe and led to an increase in the median survival of these animals. However, not all the animals responded, underscoring the need to improve this therapy. In order to increase the antitumoral effect of the virus, we have engineered Delta-24-RGD with the costimulatory ligand 4-1BBL (Delta24-ACT). 4-1BB is a costimulatory receptor that promotes the survival and expansion of activated T cells, and the generation and maintenance of memory CD8+ T cells. In this project, we evaluated the oncolytic effect of Delta24-ACT and the antitumor immune response in DIPG murine models. MATERIALS AND METHODS We use the NP53 and XFM murine DIPG cell lines. Flow cytometry was used to assess cell infectivity and ligand expression. We analyzed viral replication using a method based in hexon detection, and viral cytotoxic effect using the MTS assay. For immunogenic cell death analysis, we measured ATP secretion by a luminometric assay and calreticulin location by flow cytometry and immunofluorescence. For in vivo studies, cells and virus were injected in the pons of the mice, using the screw-guided system. RESULTS In vitro, Delta24-ACT was able to infect and induce cell death in a dose-dependent manner in murine DIPG cell lines. In addition, Delta24-ACT was able to replicate in these tumor cells and to express viral proteins. Moreover, infected cells expressed 41BBL in their membranes. Delta24-ACT could induce immunogenic cell death due to an increased secretion of ATP and calreticulin translocation to the membrane of infected cells (in no-infected cells it located in the ER), DAMPs that can trigger the immune response activation. In vivo, Delta24-ACT demonstrated to be safe in all the tested doses and was able to induce a significant increase in the median survival of the treated animals. Moreover, long-term survivors display immunological memory. CONCLUSIONS Delta24-ACT treatment led to antitumoral effect in DIPG murine cell lines in vitro. Of significance, we have demonstrated that in vivo administration of Delta24-ACT is safe and results in an enhanced antitumor effect. Future in vivo studies will explore the underlying immune mechanism of the virus.

EB10, an Anti-FLT3 Monoclonal Antibody, Selectively Targets ALL Cell Lines and Primary ALL Blasts without Interfering with Normal Hematopoiesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2744-2744 ◽  
Author(s):  
Obdulio Piloto ◽  
Patrick Brown ◽  
Li Li ◽  
Bao Nguyen ◽  
Kyu-Tae Kim ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Lines ◽  
Scid Mice ◽  
Cytotoxic Agents ◽  
Radioactive Isotopes ◽  
Leukemic Cells ◽  
Class Iii ◽  
Antibody Treatment

Abstract The class III receptor tyrosine kinase, FLT3, is expressed by &gt;90% of B-lineage acute lymphoblastic leukemias (ALL) blasts. In addition, it is expressed at extremely high levels in ALL patients with MLL-rearrangements or hyperdiploidy and sometimes mutated in these same patients. In this report, we investigated the effects of EB10, an anti-human FLT3 monoclonal antibody capable of preventing binding of FLT3 ligand (FL), on ALL cell lines and primary cells. In vitro studies, examining the ability of EB10 to inhibit FLT3 activation and downstream signaling in ALL cell lines and primary blasts, yielded variable results. In some cell lines FLT3 phosphorylation was inhibited and with it, downstream activation of pathways involving MAPK, AKT, and STAT5 phosphorylation. However, several cell lines actually exhibited FLT3 activation upon antibody treatment, possibly because of antibody-mediated receptor dimerization, and subsequent activation of downstream pathways. Nevertheless, through antibody-mediated cellular cytotoxicity (ADCC) such an antibody could still prove efficacious against leukemia cells in vivo. In fact, EB10 treatment significantly prolongs survival and/or reduces engraftment of ALL cell lines and primary ALL blasts in NOD/SCID mice. This effect might be even more pronounced in a host that was less immune compromised than are NOD/SCID mice. The leukemic cells surviving EB10 treatment in the mice were characterized by FACS analysis and found to express low levels or no FLT3. In contrast to the reduction in engraftment of human ALL primary blasts, EB10 treatment of NOD/SCID mice did not reduce engraftment of human hematopoietic CD34+ cells. Taken together, these data demonstrate that EB10 is selectively cytotoxic to ALL blasts while having little effect on normal hematopoiesis. Such an antibody, either naked or conjugated to radioactive isotopes or cytotoxic agents, may prove useful in the therapy of infant ALL as well as childhood and adult ALL patients whose blasts typically express FLT3.

The Multi-Targeted Receptor Tyrosine Kinase Inhibitor, ABT-869, Induces Apoptosis of AML Cells Both In Vitro and In Vivo.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 616-616 ◽  
Author(s):  
Deepa B. Shankar ◽  
Jenny C. Chang ◽  
Bertrand Parcells ◽  
Salemiz Sandoval ◽  
Junling Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tyrosine Kinase ◽  
Cell Line ◽  
Progenitor Cells ◽  
Cell Lines ◽  
Receptor Tyrosine Kinase ◽  
Scid Mice ◽  
Parp Cleavage

Abstract Children with acute myeloid leukemia (AML) have less than 60% overall survival despite aggressive chemotherapy and bone marrow transplantation. Only one third of the adult patients diagnosed with AML will be cured. AML blast cells from up to 30% of patients express a constitutively active receptor tyrosine kinase, FLT3-ITD, which contains an internal tandem duplication in the juxtamembrane domain. Patients with FLT3-ITD have a worse prognosis. ABT-869 is a novel multi-targeted small molecule inhibitor of receptor tyrosine kinases and is a potent inhibitor of FLT3, c-Kit, and all members of the VEGF and PDGF receptor families. To determine the effects of ABT-896 on AML cells, we treated AML cell lines, primary cells, and tumors in xenograft models with varying concentrations of the drug. In vitro viability assays showed that ABT-869 inhibited the growth of two different cell lines, MV-4-11 (human AML cell line that expresses FLT3-ITD) and BAF3-ITD (murine B-cell line stably transfected with the FLT3-ITD) at an IC50 of 10nM. ABT-869 was also effective against another mutation of FLT3, D835V, but at higher concentrations (IC50 of 100nM). Phosphorylation of FLT3 and activation of downstream signaling molecules, STAT5 and ERK, were inhibited by ABT-869 in a concentration-dependent manner. Cells were also stained with Annexin V-FITC and Propidium Iodide, and analyzed using FACS. ABT-869 induced apoptosis, caspase-3 activation, and PARP cleavage after 48 hours. To examine the in vitro effects of ABT-869 on normal hematopoietic progenitor cells, we performed methylcellulose-based colony assays with human bone marrow. No significant difference was observed in the number and type of colonies formed using BM cells treated with ABT-869 or control, up to a concentration of 1 micromolar. These results suggest that ABT-869 is not toxic to normal bone marrow progenitor cells at concentrations that are effective against AML cells. To examine the effects of ABT-869 in vivo, we treated SCID mice injected with MV-4-11, Baf3-ITD, Baf3-D835V, or Baf3-WT cells, with oral preparations of ABT-869. Complete regression of MV-4-11 tumors was observed in mice treated with ABT-869 at 20 and 40 mg/kg/day. No adverse effects were detected in the peripheral blood counts, bone marrow, spleen or liver. Histology of the tumors from the control-treated group showed a high degree of proliferation by Ki-67 staining, increased mitotic figures, and a well-defined tumor mass. In contrast, the tumors from mice treated with ABT-869 showed a number of apoptotic bodies by TUNEL staining and the presence of reactive, inflammatory cells. Interestingly, we also observed that mice that received ABT-869 the day after injection of AML cells remained tumor-free for over 2 months in contrast to the mice receiving the vehicle alone. Inhibition of FLT3 phosphorylation was demonstrated in the tumors from mice treated with ABT-869. We are evaluating the activity of ABT-869 treatment of SCID mice injected with Baf3-ITD, Baf3-D835V, or Baf3-WT cells. NOD-SCID mouse models are currently being used to analyze the effects of ABT-869 on primary AML cells in vivo. Our preclinical studies demonstrate that ABT-869 is effective and nontoxic, and provide rationale for the treatment and prevention of relapse in AML patients.

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